DNA damage response in a 2D-culture model by diffusing alpha-emitters radiation therapy (Alpha-DaRT)

Diffusing alpha-emitters radiation therapy (Alpha-DaRT) is a unique method, in which interstitial sources carrying 224Ra release a chain of short-lived daughter atoms from their surface. Although DNA damage response (DDR) is crucial to inducing cell death after irradiation, how the DDR occurs during Alpha-DaRT treatment has not yet been explored. In this study, we temporo-spatially characterized DDR such as kinetics of DNA double-strand breaks (DSBs) and cell cycle, in two-dimensional (2D) culture conditions qualitatively mimicking Alpha-DaRT treatments, by employing HeLa cells expressing the Fucci cell cycle-visualizing system. The distribution of the alpha-particle pits detected by a plastic nuclear track detector, CR-39, strongly correlated with γH2AX staining, a marker of DSBs, around the 224Ra source, but the area of G2 arrested cells was more widely spread 24 h from the start of the exposure. Thereafter, close time-lapse observation revealed varying cell cycle kinetics, depending on the distance from the source. A medium containing daughter nuclides prepared from 224Ra sources allowed us to estimate the radiation dose after 24 h of exposure, and determine surviving fractions. The present experimental model revealed for the first time temporo-spatial information of DDR occurring around the source in its early stages.

Appendix: Estimating the mean number of hits to the cell nucleus and the absorbed dose from the density of recorded CR-39 etch pits 1. Estimating the fraction of alpha particles hitting CR-39 which create detectable etch pits Only a fraction of the alpha particles hitting the CR-39 film create detectable etch pits.
To create detectable pits, two conditions must be met: (1) the angle of the alpha particle track with respect to the CR-39 surface must be larger than an energydependent critical angle; (2) the penetration depth of the track into the CR-39 film must be larger than the thickness of the etched layer.
The dependence of the critical angle on the energy of the alpha particle as it hits the CR-39 surface is commonly parametrized through a sensitivity function ("V function") which depends on the residual range ' of the alpha particle in CR-39: There are several published parametrization forms of (').Here we use a form by Hermsdorf 1 : with  # = 390 ,  -= 2 m,  * = 1 m,  .= 5 m,  , = 80 m,  # = 2.35.
2. 10 8 alpha particle tracks were sampled from isotropic sources (emitting into a solid angle of 4π) with an initial height above the CR-39 plane  & sampled from a uniform distribution between 0 and  !"#$ (H % O).
3. For each alpha particle reaching  = 0 we calculated the residual energy ' (after traversing a path length  =  & /cos θ, with  being the emission angle relative to the negative z axis (0 ≤ θ ≤ π/2).The residual range ' in CR-39 corresponding to the residual energy ' was found by interpolating on tabulated data from SRIM 2013 2 .
4. An alpha particle was considered to be detected if the angle of its track relative to the CR-39 surface was larger than the critical angle, and if the depth of its penetration into the CR-39 film,  ()*+, = 'cosθ was larger than the thickness of the etched layer (6.9 μm).

Estimating the number of alpha hits per nucleus
The number of alpha hits per nucleus was estimated in a second in-house Monte Carlo simulation (implemented in MATLAB) as follows.The nucleus was simulated as an oblate spheroid with a horizontal radius  = 9.44 ± 0.66 µm (corresponding to a cross section area of 280 ± 39 μm 2 ) and a vertical radius  = 5 ± 1 µm, with its center at  = .Alpha particle tracks were generated randomly in a cylindrical domain surrounding the nucleus with a radius  28: =  +  and height  28: = 2 + , where  was set separately to the alpha particle CSDA range of 212 Bi and 212 Po.The alpha particle emission points were sampled uniformly throughout the cylindrical domain (including inside the nucleus) with isotropic directions.The calculation was performed separately for 212 Bi and 212 Po, counting in each case the number of straight tracks crossing the nucleus surface at least once.The ratio between the number of nucleus hits and number density of alpha decays (number of simulated decays divided by the domain volume) is given in Table 2.The uncertainties were estimated by sampling the spheroid radii randomly from normal distributions with their respective standard deviations.The MC simulation employed for estimating the mean number of alpha hits to the nucleus was further used to calculate the absorbed dose.For tracks crossing the nucleus surface at least once, we scored the energy deposited in the nucleus  23; using / data taken from the NIST ASTAR online database (https://physics.nist.gov/PhysRefData/Star/Text/ASTAR.html).The specific energy for the i-th track was calculated as  0 =  23; 0 / <=! , where  <=! is the nucleus mass (calculated assuming a medium density  = 1 g/cm 3 ).The total number of tracks for decays/μm 3 , and the volume of the cylindrical domain surrounding the nucleus (described above).This allowed scoring the total energy deposited in the nucleus  = ∑  0 for a run simulating the real experiment.The process was repeated 10 4 times to produce the statistical distribution of the total specific energy ().The absorbed alpha dose was then taken as average value of the distribution of the total specific energy,  > = ̅ = ∫ ()  .Using this methodology, for the most active DM (dilution 1/2×) we estimated an alpha dose of 1.10 ± 0.23 Gy.
The absorbed beta dose was grossly estimated as follows.For an infinite medium with a uniform concentration of For the most active DM (with  23!$45 ( α) = (5.32 ± 1.11) ⋅ 10 ". decays/μm 3 ) the "infinite medium" beta dose was calculated to be 0.19 ± 0.04 Gy.On the boundary between two infinite media, one with a uniform density of beta emitters and one with zero activity, the beta dose is precisely half of that in an infinite medium.As a very rough estimate, we adopted this value for the absorbed beta dose experienced by the cell, giving  ?() ≈ 0.09 Gy.Considering any reasonable value of RBE for alpha particles, and an alpha particle dose of 1.1 Gy, the relative beta contribution to cell survival can be expected to be at most on the few % level.